Advances in our understanding of T cell activation at a molecular level have permitted the manipulation of T cell regulation in cancer patients. An important molecule involved in T cell regulation is CD40 which is expressed on antigen-presenting cells and B cells. The CD40-CD40 ligand interaction is an essential element in the generation of T cell help which maintains potent, high-avidity and long lasting memory responses. A human agonistic CD40 antibody has been shown in animal tumor models and in melanoma patients to have significant anti-tumor activity and has shown potent anti-tumor effects in mice in combination with TLR agonists, especially TLR3 agonist poly IC:LC. Experiments with murine PBMC indicate that CD40 antibody with TLR3 agonist increases the generation of antigen specific T cells that are lytic, functional, gamma-interferon secreting effector cells. When CD40 agonistic antibody is combined with TLR3 agonist poly IC:LC in vivo in animal models there is an additive or even a synergistic anti- tumor effect. Based on those pre-clinical data, and preliminary clinical evidence of anti-tumor activity of CD-40 antibody, we propose to perform a pilot escalating dose trial in resected high- risk stages III/ IV melanoma patients of a multi-peptide vaccine and poly IC:LC with placebo or at least two different doses of anti-CD40 agonistic antibody in cohorts of 10 patients each. The endpoints will be toxicity, definition of an MTD and a comparison of immune responses between the cohorts as measured by different functional T cell assays. The hypothesis being tested is that a CD40 agonistic antibody when combined with a TLR3 agonist and a melanoma vaccine results in a dose-dependent augmentation in the generation of highly avid, long lived memory- effector T cells that are antigen specific. PUBLIC HEALTH RELEVANCE: In the clinical trial that this proposal will support, patients with resected stages III and IV melanoma will have the opportunity to be treated with a regimen that has, in our opinion, a significant chance of benefiting them. The conduct of this trial will provide important information that may help patients with resected melanoma that have a very high (80% or more) risk of death from melanoma, and provide a framework to test new and promising vaccines that might benefit other patients with cancer. The general health of all Americans would be improved if we could understand the mechanisms by which T cell immunity with high-avidity, long lasting anti-tumor effector cells were generated in cancer patients.